This invention relates to high level microbial production of swine growth hormone through recombinant DNA technology. This high level production is achieved through high-density fermentation of E. coli cells transformed with a recombinant vector carrying a gene encoding swine growth hormone.
Swine growth hormone (SGH) is a protein of 190 amino acids, which is initially synthesized in the anterior pituitary gland as a precursor "pre-growth hormone" having 26 additional amino acids attached at the N-terminus. This 26-amino acid "signal sequence" is processed off during secretion from the pituitary cells, yielding the mature hormone. It has been demonstrated that administration of SGH, purified from pituitary glands, to pigs results in increased growth rates, improved feed-to-animal weight conversion, improved meat quality in terms of nutritional content, and improved carcass quality in terms of increased length and reduced backfat. (See, for example, European Patent Application Publication No. 0 104 920.) The potential economic value of this hormone sparked interest in obtaining SGH in commercial quantities at reasonable cost.
Thus, much work in recent years has focused on obtaining microbial synthesis of this commercially valuable hormone using recombinant DNA technology. Gene cloning and manipulation techniques well known in the art have been used to produce recombinant expression vectors containing SGH-encoding cDNA fused to regulatory regions capable of directing synthesis of SGH in the desired host cells. Microorganisms transformed with these expression vectors have been shown to produce the desired hormone. For example, the cloning of SGH-specific cDNa and construction of expression vectors therefrom is described in European Patent Application Publication No. 0 104 920 (hereinafter referred to as EPO No. 0 104 920). The highest yield of SGH reported in this publication, 32 mg/liter, was achieved in small scale cultures of E. coli cells transformed with both an expression vector encoding .DELTA.-7 SGH (an SGH polypeptide lacking the first seven amino-terminal amino acids of the mature hormone) and a plasmid carrying a gene encoding a temperature-sensitive repressor to control SGH synthesis. Large-scale fermentation of any of the transformed strains is not reported. Seeburg et al., (DNA, 2:37-45 [1983]) describe the cloning of bovine and porcine (swine) growth hormone cDNA and construction of expression vectors encoding the complete mature hormones (i.e., the "pre" or signal sequence region is removed in vitro during vector construction). E. coli cells were transformed with the SGH expression vector, and SGH synthesis was regulated by the plasmid-borne E. coli trp regulatory region. It is reported that high density fermentation of the transformed E. coli cells yielded approximately 1.5 grams/liter SGH, but no description of the fermentation conditions is given.
Obtaining maximal expression levels of the protein products of cloned genes often involves some trial and error. The genes may be fused to several different regulatory regions and/or transformed into several host cell strains for comparative analyses to find the transformed strain giving the highest production levels of the desired protein. To date, efforts at yield improvement of microbially produced growth hormones have been carried out primarily at the level of genetic manipulations designed to increase cellular expression. There is still a need for the development of commercial scale fermentation processes capable of producing growth hormones in the highest possible yields.